AcidSensor Labeling Kit – Endocytic Internalization Assay

04 Intracellular Fluorescent Probes

AcidSensor Labeling Kit – Endocytic Internalization Assay

AcidSensor Labeling Kit – Endocytic Internalization Assay

pH Sensor Labeling Kit

  • Label with pH-sensitive fluorescent dye
  • All-in-one Kit
  • Detailed Labeling Manual
  • Product code
    A558　 AcidSensor Labeling Kit – Endocytic Internalization Assay
Unit size Price Item Code
3 samples ＄380.00 A558-10
Amount of Sample 50-200 µg
Labeling Group -NH2
Instrument Microscope/ FCM
Fluorescence [Ex:645, Em:666]

・Proteins with a molecular weight above 50,000 can be labeled.

・High recovery rate using a filtration tube.

Order
3 samples
Quantity may be adjusted within the cart.
Component
3 samples ・NH2-Reactive AcidSensor
・WS Buffer
・Reaction Buffer
・Filtration Tube		 3 tubes
4 ml×1
500 μl×1
3 tubes

Description

This kit is an all-in-one kit that allows visualization of the endocytosis uptake of a target substance. The NH2-Reactive AcidSensor (fluorescent probe) included in the kit has an intramolecular active ester group that forms a stable covalent bond when mixed with an amino group-containing target substance (protein). The AcidSensor label can be excited at 633 nm, allowing for multiple staining with green or red fluorescence (Figure 1). The AcidSensor label shows little fluorescence in neutral conditions and fluoresces when acidified in the cells where it is taken up by endocytosis (Figure 2).*Notice:
・Unlike the endocytosis detection dye: ECGreen (product code: E296), this kit stains target substances that enter the cell.
・This kit can label samples with molecular weights of more than 50,000 and with reactive amino groups.

Manual

Technical info

This kit includes a filtration tube necessary to remove the unreacted dye, and allows you to perform everything from labeling to purification operations. *
In addition, even first-time users can easily label AcidSensor by conducting experiments according to the instruction manual.

* Protein/Antibody is not included.
* Please prepare DMSO and a proper medium.

Compare with other Kits

 

 

Co-staining for Observation with Endosomes

Cellular uptake of labeled IgG over time 

AcidSensor-labeled mouse IgG stained with this kit and Dojindo's endocytosis detection dye （Code: E296, ECGreen -Endocytosis Detection）were added to HeLa cells. Observed the cells 10, 20, and 180 minutes after the staining, the results showed that the AcidSensor (Deep Red) and endosomal membranes(Green) were localized at the same place, indicating that mouse IgG was uptake by the cells via the endocytosis pathway. 


＜Observation Conditions＞
Green：ECGreen (Ex = 405 nm, Em = 500-550 nm)
Deep Red：AcidSensor (Ex = 633 nm, Em = 650-700 nm)

Application Data AcidSensor Labeled BSA and Mouse IgG Uptake by HeLa Cells

Labeled BSA or Mouse IgG was added to HeLa cells using this kit, and uptake by HeLa cells was observed after 2 hours. As a result, AcidSensor fluorescent was detected in HeLa cells, confirming that each labeled object was taken up by the endocytosis pathway.


＜Observation Conditions＞
Instrument: Confocal Microscope
Ex = 633 nm, Em = 650-700 nm

Q & A

Q

Do coexisting substances in the sample solution affect the labeling reaction?

A

Depends on the coexisting substances.
After confirming what substances are contained in the solution, purify the sample accordingly.
Coexisting compounds with amino groups and a molecular weight above 10,000 will decrease the labeling efficiency, and cannot be removed. Even for compounds without amino groups, high molecular weight impurities can cause clogging of the filter, which may interfere with labeling and purification. Please purify the sample before using it.

Q

Is there any cytotoxicity in the WS buffer？

A

WS buffer contains a detergent in a quantity that shows almost no cytotoxicity. If you are concerned about the effect on cells, collect the labeled substance using a buffer of your choice.

Q

Is it possible to take up or observe a labeled substance in serum-containing medium?

A

Serum components do not affect AcidSensor performance, but serum proteins may inhibit the uptake of the endocytosis pathway. Please consider whether to include serum or not depending on your experiment.

Q

How should I calculate the labeling rate?

A

Dilute the labeled sample 5-fold with WS buffer, measure the absorbance at 500 nm and 280 nm, and calculate the labeling rate using the following formula.

A500: Absorbance at 500 nm 

A280: Absorbance at 280 nm

εprotein: The proteins' coefficient of molar absorbance at 280nm

The A500 coefficient of molar absorbance of NH2-Reactive AcidSensor is  48400

Using the A280 and A 500 of NH2-Reactive AcidSensor, the ratio of 280nm/500nm is 0.16

For IgG samples, the εprotein is 216,000

Q

How many NH2-Reactive AcidSensors are labeled per protein molecule?

A

For Mouse IgG, we have confirmed that an average of three AcidSensors are labeled per molecule.

Handling and storage condition

Handling and storage condition
Store in a cool (0–5°C), dark, and dry place.
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